The role of site-specific N-glycosylation in secretion of soluble forms of rabies virus glycoprotein.
Rabies virus glycoprotein is important in the biology and pathogenesis of neurotropic rabies virus infection. This transmembrane glycoprotein is the only viral protein on the surface of virus particles, is the viral attachment protein that facilitates virus uptake by the infected cell, and is the target of the host humoral immune response to infection. The extracellular domain of this glycoprotein has N-glycosylation sequons at Asn37, Asn247, and Asn319. Appropriate glycosylation of these sequons is important in the expression of the glycoprotein. Soluble forms of rabies virus glycoprotein were constructed by insertion of a stop codon just external to the transmembrane domain. Using site-directed mutagenesis and expression in transfected eukaryotic cells, it was possible to compare the effects of site-specific glycosylation on the cell-surface expression and secretion of transmembrane and soluble forms, respectively, of the same glycoprotein. These studies yielded the surprising finding that although any of the three sequons permitted cell surface expression of full-length rabies virus glycoprotein, only the N-glycan at Asn319 permitted secretion of soluble rabies virus glycoprotein. Despite its biological and medical importance, it has not yet been possible to determine the crystal structure of the full-length transmembrane form of rabies virus glycoprotein which contains heterogeneous oligosaccharides. The current studies demonstrate that a soluble form of rabies virus glycoprotein containing only one sequon at Asn319 is efficiently secreted in the presence of the N-glycan processing inhibitor 1-deoxymannojirimycin. Thus, it is possible to purify a conformationally relevant form of rabies virus glycoprotein that contains only one N-glycan with a substantial reduction in its microheterogeneity. This form of the glycoprotein may be particularly useful for future studies aimed at elucidating the three-dimensional structure of this important glycoprotein.
Wojczyk
B S
BS
Department of Pathology and Laboratory Medicine, Wistar Institute, University of Pennsylvania, Philadelphia 19104, USA.
Stwora-Wojczyk
M
M
Shakin-Eshleman
S
S
Wunner
W H
WH
Spitalnik
S L
SL
D000818:Animals; D000956:Antigens, Viral / Q000096:biosynthesis / Q000276:immunology / Q000378:metabolism; D016466:CHO Cells; D002352:Carrier Proteins / Q000378:metabolism; D002460:Cell Line; D006224:Cricetinae; D000091342:Endoplasmic Reticulum Chaperone BiP; D006023:Glycoproteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D006031:Glycosylation / Q000187:drug effects; D006360:Heat-Shock Proteins; D007212:Indolizines / Q000494:pharmacology; D007424:Intracellular Fluid / Q000378:metabolism; D007700:Kinetics; D018832:Molecular Chaperones / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D011820:Rabies virus / Q000235:genetics / Q000276:immunology / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000138:chemical synthesis / Q000187:drug effects / Q000378:metabolism; D012995:Solubility; D017026:Swainsonine / Q000494:pharmacology; D014162:Transfection; D014759:Viral Envelope Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D054836:Indolizidines
D000956:Antigens, Viral; D002352:Carrier Proteins; D000091342:Endoplasmic Reticulum Chaperone BiP; D006023:Glycoproteins; D006360:Heat-Shock Proteins; D007212:Indolizines; D018832:Molecular Chaperones; D011993:Recombinant Fusion Proteins; D017026:Swainsonine; D014759:Viral Envelope Proteins; C064951:glycoprotein G, Rabies virus; C037806:castanospermine; D054836:Indolizidines